Methods for treating tourniquet-induced injuries

ABSTRACT

The present invention relates to treating a tissue in a mammal from the effects of reperfusion using flagellin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/530,580, filed Jun. 22, 2012, now U.S. Pat. No. 8,618,059, which is acontinuation of U.S. patent application Ser. No. 13/056,973, filed Jan.31, 2011, now U.S. Pat. No. 8,324,163, which is a 371 nationalapplication of International Patent Application No. PCT/US2009/052493,which claims the benefit of U.S. Provisional Application No. 61/085,766,filed on Aug. 1, 2008, the contents of all of which are incorporatedherein by reference.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:CLEV_(—)008_(—)03US_SeqList_ST25.txt, date recorded: Mar. 24, 2014, filesize 135 kilobytes).

FIELD OF THE INVENTION

This invention relates to the use of flagellin related polypeptides totreat tissues from the effects of reperfusion.

BACKGROUND OF THE INVENTION

Tissues deprived of blood and oxygen undergo ischemic necrosis orinfarction with possible irreversible organ damage. Once the flow ofblood and oxygen is restored to the organ or tissue (reperfusion), theorgan does not immediately return to the normal preischemic state.Reperfusion of coronary flow is necessary to resuscitate the ischemic orhypoxic tissue or organ. Timely reperfusion facilitates salvage of cellsand decreases morbidity and mortality. Reperfusion of an ischemic areamay result in a paradoxical dysfunction including marked endothelialcell dysfunction, which results in vasoconstriction, platelet andleukocyte activation, increased oxidant production, and increased fluidand protein extravasation.

Over the past two decades has witnessed several pharmacologicalinterventions designed to limit reperfusion injury. Unfortunately, thesuccess of some agents has been limited to experimental model ofischemia and reperfusion. The lack of consistent clinical benefit may berelated to a variety of factors, including poor clinical trial design,inadequate pharmacokinetic/pharmacodynamic studies and the complexity ofthe human in vivo model.

There is a need in the art to distinguish therapeutic strategies forischemia vs. reperfusion, and it is possible that a combination ofagents is required to elicit the maximum clinical benefit.

SUMMARY OF THE INVENTION

Provided herein is a method of treating a tissue of a mammal from theeffects of reperfusion, which may comprise administering to a mammal inneed thereof a composition comprising flagellin. The composition may beadministered in combination with an antioxidant, which may be selectedfrom the group consisting of amifostine and vitamin E.

The reperfusion may be caused by an injury, which may be ischemia orhypoxia. The ischemia may be selected from the group consisting oftachycardia, infarction, hypotension, embolism, thromboemoblism (bloodclot), sickle cell disease, localized pressure to extremities to thebody, and tumors. The hypoxia may be selected from the group consistingof hypoxemic hypoxia (carbon monoxide poisoning; sleep apnea, chronicobstructive pulmonary disease, respiratory arrest; shunts), anemichypoxia (O2 content low), hypoxemic hypoxia, and histotoxic hypoxia. Thelocalized pressure may be due to a tourniquet.

The composition may be administered prior to, together with, or afterthe influx of oxygen. The tissue may be selected from the groupconsisting of GI tract, lung, kidney, liver, cardiovascular system,blood vessel endothelium, central nervous system, peripheral nervoussystem, muscle, bone, and hair follicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the level of creatinine in the serum of mice over 5days post intravenous administration of flagellin at concentrations ofeither 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg/body.

FIG. 2 demonstrates the affect of flagellin administered to mice beforeimposition of renal ischemia and measuring survival and creatininefollowing reperfusion of the ischemic kidneys. Panel A shows the percentsurvival of mice who were pretreated with flagellin at concentrations ofeither 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg/body or PBS as a control.Panel B shows the level of creatinine in the same group of pretreatedand control mice.

FIG. 3 demonstrates the histopathology of ischemic kidney cells 24 hoursafter reperfusion that were pretreated with PBS or flagellin atconcentrations of either 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg/body. TheSham column indicates kidney cells isolated from mice that were notimposed with renal ischemia.

FIG. 4 demonstrates histopathology of kidney cells 7 days afterreperfusion. In the first panel, the histopathology slide shows kidneycells isolated from mouse pretreated with PBS before renal ischemia andfollowed by reperfusion of the ischemic kidneys. In the first panel, thehistopathology slide shows kidney cells isolated from mouse pretreatedwith PBS before renal ischemia and followed by reperfusion of theischemic kidneys. In the second panel, the histopathology slide showskidney cells isolated from mouse pretreated with flagellin at aconcentration of 0.5 μg/body but not imposed with renal ischemia. Thethird panel demonstrates a histopathology slide showing kidney cellsisolated from a mouse pretreated with flagellin at a concentration of0.5 μg/body and imposed with renal ischemia and followed by reperfusionof the ischemic kidneys.

FIGS. 5 a-5 c demonstrate assessing leukocyte infiltration 9 hours and24 hours after reperfusion into ischemic kidney cells isolated from micepretreated with PBS or flagellin at 0.5 μg/body. FIG. 5 a is kidneytissue cells immunohistochemically stained for levels of neutrophilinfiltration 9 hours and 24 hours after reperfusion in ischemic andnon-ischemic treated kidneys cells from mice pretreated with PBS orflagellin at 0.5 μg/body. FIG. 5 b is the number of neutrophils,macrophages, CD4⁺ T cells, and CD8⁺ T cells infiltrating into kidneytissue cells isolated from mice pretreated with PBS or flagellin aconcentration of 0.5 μg/body. FIG. 5 c shows the effects of flagellin onmyeloperoxidase levels, as a measure of the activation of neutrophils,during reperfusion of ischemic kidneys as compared to controls.

FIG. 6 a demonstrates critical role of flagellin preventing chemokinesCXCL1/KC and CXCL2/KC in directing leukocyte infiltration into ischemickidney tissues. FIG. 6 b demonstrates mRNA levels of the acute phaseproteins IL-1b and IL-6 but not TNFa were also decreased in ischemickidneys at 9 hours post-reperfusion in flagellin preconditioned animals.

FIG. 7 demonstrates survival and creatinine levels in groups of C57BL/6mice that were subjected to 45 minutes of bilateral renal pedicleocclusion and were administered 0.5 μg of flagellin at various timesfollowing the removal of the renal clamps. FIG. 7 a shows the effects ofadministering flagellin 30 minutes before or within 30 minutes afterdeclamping on the viability of mice subjected to ischemic injury. FIG. 7b shows the effects of administering flagellin 30 minutes beforedeclamping or within 30 minutes following declamping on levels of serumcreatinine after reperfusion of the ischemic kidneys. FIG. 7 c shows theeffects of flagellin on CXCL1 and CXCL2 levels at 9 and 24 hours afterreperfusion.

FIG. 8 demonstrates administration of 0.5 μg flagellin within 30 minutesof reperfusion of ischemic kidneys of wild-type C57BL/6 micereconstituted with wild-type bone marrow decreased CXCL1 and CXCL2 mRNAlevels. In MyD88^(−/−) recipients reconstituted with either MyD88^(−/−)or wild-type bone marrow, little CXCL1 and CXCL2 mRNA was induced duringreperfusion of ischemic kidneys and administration of flagellin duringreperfusion of these kidneys did not decrease the mRNA levels of thesechemokines. In contrast, wild-type recipients of bone marrow fromMyD88^(−/−) donors expressed high levels of CXCL1 and CXCL2 mRNA andthese levels were decreased by administration of flagellin duringreperfusion of the ischemic kidneys.

FIG. 9 a demonstrates renal sections from wild-type C57BL/6 and BALB/cmice were stained with anti-TLR5 antibody. FIG. 9 b demonstratesexpression levels of TLR5 mRNA were low in kidneys prior to impositionof renal ischemia/reperfusion but increased quickly during reperfusionof ischemic kidneys.

FIG. 10 shows the domain structure of bacterial flagellin. The Cabackbone trace, hydrophobic core distribution and structural informationof F41. Four distinct hydrophobic cores that define domains D1, D2a, D2band D3. All the hydrophobic side-chain atoms are displayed with the Cabackbone. Side-chain atoms are color coded: Ala, yellow; Leu, Ile orVal, orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms).c, Position and region of various structural features in the amino-acidsequence of flagellin. Shown are, from top to bottom: the F41 fragmentin blue; three b-folium folds in brown; the secondary structuredistribution with a-helix in yellow, b-structure in green, and b-turn inpurple; tic mark at every 50th residue in blue; domains D0, D1, D2 andD3; the axial subunit contact region within the proto-element in cyan;the well-conserved amino-acid sequence in red and variable region inviolet; point mutations in F41 that produce the elements of differentsupercoils. Letters at the bottom indicate the morphology of mutantelements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V),R-type straight; C (D313Y, A414V, A427V, N433D), curly33.

FIG. 11 shows a schematic of Salmonella flagellin domains, itsfragments, and its interaction with TLR5. Dark bars denote regions ofthe flagellin gene used to construct fragments comprising A, B, C, A′and B′.

FIG. 12 depicts flagellin derivatives. The domain structure andapproximate boundaries (amino acid coordinates) of selected flagellinderivatives (listed on the right). F11C flagellin of Salmonella dublinis encoded within 505 amino acids (aa).

FIGS. 13A-K show the nucleotide and amino acid sequence for thefollowing flagellin variants: AA′ (SEQ ID NO: 7-8), AB′ (SEQ ID NO:9-10), BA′ (SEQ ID NO: 11-12), BB′ (SEQ ID NO: 13-14), CA′ (SEQ ID NO:15-16), CB′ (SEQ ID NO: 17-18), A (SEQ ID NO: 19-20), B (SEQ ID NO:21-22), C (SEQ ID NO: 23-24), GST-A′ (SEQ ID NO: 25-26), GST-B′ (SEQ IDNO: 27-28), AA′n1-170 (SEQ ID NO: 29-30), AA′n1-163 (SEQ ID NO: 33-34),AA′n54-170 (SEQ ID NO: 31-32), AA′n54-163 (SEQ ID NO: 335-36), AB′n1-170(SEQ ID NO: 37-38), AB′n1-163 (SEQ ID NO: 39-40), AA′n1-129 (SEQ ID NO:41-42), AA′n54-129 (SEQ ID NO: 43-44), AB′n1-129 (SEQ ID NO: 45-46),AB′n54-129 (SEQ ID NO: 47-48), AA′n1-100 (SEQ ID NO: 49-50), AB′n1-100(SEQ ID NO: 51-52), AA′n1-70 (SEQ ID NO: 53-54) and AB′n1-70 (SEQ ID NO:55-56). The pRSETb leader sequence is shown in Italic (leader includesMet, which is also amino acid 1 of FliC). The N terminal constant domainis underlined. The amino acid linker sequence is in Bold. The C terminalconstant domain is underlined. GST, if present, is highlighted.

FIG. 14A shows histological section of mice hind limb muscle 14 daysafter reperfusion following 3 hours of warm ischemia using ahematoxylin/eosin stain where the mouse had been given 0.5 μg of CBLB502within 15 minutes of reperfusion. FIG. 14B shows histological section ofmice hind limb muscle 14 days after reperfusion following 3 hours ofwarm ischemia using a hematoxylin/eosin stain where the mouse had beengiven vehicle (PBS) within 15 minutes of reperfusion. FIG. 14C shows thewet/dry ratio of tissue edema in the limb of mice administered eitherwith CBLB502 or PBS within 15 minutes of reperfusion after 3 hours ofischemia. The ratio of edema was also measured in the limb of miceadministered CBLB502 or PBS, but spared 3 hours of ischemia. FIG. 14Dshows the wet/dry ration of vascular leaks using Blue Dye per gramweight limb of mice administered either CBLB502 or PBS within 15 minutesof reperfusion after 3 hours of ischemia. The ratio of vascular leakswas also measured in the limb of mice administered CBLB502 or PBS, butspared 3 hours of ischemia.

FIGS. 15A-C show a comparison of amino acid sequences of the conservedamino (FIG. 15A) and carboxy (FIGS. 15B and 15C) terminus from 21species of bacteria. The 13 conserved amino acids important for TLR5activity are shown with shading. The amino acid sequences are identifiedby their accession numbers from TrEMBL (first letter=Q) or Swiss-Prot(first letter=P). The amino terminus sequences have SEQ ID NOs: 57-77,respectively, for each of the 21 bacterial species, and the carboxyterminus sequences have SEQ ID NOs: 78-98, respectively.

DETAILED DESCRIPTION

The inventors have made the surprising discovery that flagellin protectsfrom the effects of reperfusion. The absence or reduction of oxygen andnutrients from blood creates a condition in which the restoration ofcirculation results in inflammation and oxidative damage through theinduction of oxidative stress rather than the restoration of normalfunction. The restored blood flow reintroduces oxygen within cells thatdamages cellular proteins, DNA and the plasma membrane. Damage to cell'smembrane may in turn cause the release of more free radicals. Suchreactive species also act in redox signaling to induce apoptosis ofischemic tissue cells. In addition, inflammatory response furtherdamages the tissue. White blood cells carried to the area by the newlyreturning blood release a host of inflammatory factors such asinterleukins as well as free radicals in response to tissue damage.Leukocytes may also build up in small capillaries, obstructing them andleading to more ischemia. While not being bound by theory, flagellin mayprovide protection from the effects of reperfusion by reducing theoxidative and inflammatory stresses to the tissue thereby preventingapoptosis and allowing faster recovery of the tissue to a normal state.This protective nature of flagellin can either by used at the onset ofreperfusion or be used to prevent further damage due to reperfusion. Thebelow-described invention relates in part to administration of flagellinto a treat tissue of a mammal from the effects of reperfusion.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

“Administer” may mean a dosage of an agent that induces NF-κB activity,means a single dose or multiple doses of the agent.

“Analog” may mean, in the context of a peptide or polypeptide, a peptideor polypeptide comprising one or more non-standard amino acids or otherstructural variations from the conventional set of amino acids.

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, fragments or derivatives thereof, including Fab, F(ab′)₂, Fd,and single chain antibodies, diabodies, bispecific antibodies,bifunctional antibodies and derivatives thereof. The antibody may be amonoclonal antibody, polyclonal antibody, affinity purified antibody, ormixtures thereof which exhibits sufficient binding specificity to adesired epitope or a sequence derived therefrom. The antibody may alsobe a chimeric antibody. The antibody may be derivatized by theattachment of one or more chemical, peptide, or polypeptide moietiesknown in the art. The antibody may be conjugated with a chemical moiety.

“Apoptosis” may mean a form of cell death that includes progressivecontraction of cell volume with the preservation of the integrity ofcytoplasmic organelles; condensation of chromatin (i.e., nuclearcondensation), as viewed by light or electron microscopy; and/or DNAcleavage into nucleosome-sized fragments, as determined by centrifugedsedimentation assays. Cell death occurs when the membrane integrity ofthe cell is lost (e.g., membrane blebbing) with engulfment of intactcell fragments (“apoptotic bodies”) by phagocytic cells.

A “peptide” or “polypeptide” may mean a linked sequence of amino acidsand may be natural, synthetic, or a modification or combination ofnatural and synthetic.

“Treating,” “treatment,” or “to treat” each may mean to alleviate,suppress, repress, eliminate, prevent or slow the appearance ofsymptoms, clinical signs, or underlying pathology of a condition ordisorder on a temporary or permanent basis. Preventing the diseaseinvolves administering a composition of the present invention to ananimal prior to onset of the disease. Suppressing the disease involvesadministering a composition of the present invention to an animal afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering a composition of the presentinvention to an animal after clinical appearance of the disease.

2. TREATING THE EFFECTS OF REPERFUSION

Provided herein is a method of treating the effects of reperfusion byadministering to a mammal in need thereof a composition comprisingflagellin. Reperfusion may be caused by an injury.

Reperfusion may damage a body component when blood supply returns to thebody component after the injury. The effects of reperfusion may be moredamaging to the body component than the injury itself. There are severalmechanism and mediators of reperfusion including oxygen free radicals,intracellular calcium overload, and endothelial dysfunction. Excessivequantities of reactive oxygen species, when reintroduced into apreviously injured body component, undergo a sequential reductionleading to the formation of oxygen free radicals. Potent oxidantradicals, such as superoxide anion, hydroxyl radical, and peroxynitritemay be produced within the first few minutes of reflow to the bodycomponent and may play a crucial role in the development of reperfusioninjury. Oxygen free radicals also can be generated from sources otherthan reduction of molecular oxygen. These sources include enzymes, suchas xanthine oxidase, cytochrome oxidase, and cyclooxygenase, and theoxidation of catecholamines.

Reperfusion is also a potent stimulus for neutrophil activation andaccumulation, which in turn serve as potent stimuli for reactive oxygenspecies production. Specifically, the main products of the neutrophilrespiratory burst are strong oxidizing agents including hydrogenperoxide, free oxygen radicals and hypochlorite. Neutrophils are themost abundant type of phagocyte, normally representing 50 to 60% of thetotal circulating leukocytes, and are usually the first cells to arriveat the site of injured body component. Oxygen-derived free radicalsproduce damage by reacting with polyunsaturated fatty acids, resultingin the formation of lipid peroxides and hydroperoxides that damage thebody component and impair the function of membrane-bound enzyme systems.Free radicals stimulate the endothelial release of platelet activatingfactor and chemokines such as neutrophil activator factor, chemokine(C—X—C motif) ligand 1, and chemokine (C—X—C motif) ligand 1 whichattracts more neutrophils and amplifies the production of oxidantradicals and the degree of reperfusion injury. Reactive oxygen speciesalso quench nitric oxide, exaggerating endothelial injury and tissuecell dysfunction. In addition to an increased production, there is alsoa relative deficiency in endogenous oxidant scavenging enzymes, whichfurther exaggerates free radical-mediated cardiac dysfunction.

Reperfusion may further result in marked endothelial cell dysfunction.Endothelial dysfunction facilitates the expression of a prothromboticphenotype characterized by platelet and neutrophil activation, importantmediators of reperfusion. Once neutrophils make contact with thedysfunctional endothelium, they are activated, and in a series ofwell-defined steps (rolling, firm adherence, and transmigration) theymigrate into areas of tissue injury through endothelial cell junctionsas part of the innate immune response.

Changes in intracellular calcium homeostasis play an important role inthe development of reperfusion. Reperfusion may be associated with anincrease in intracellular calcium; this effect may be related toincreased sarcolemmal calcium entry through L-type calcium channels ormay be secondary to alterations in sarcoplasmic reticulum calciumcycling. In addition to intracellular calcium overload, alterations inmyofilament sensitivity to calcium have been implicated in reperfusion.Activation of calcium-dependent proteases (calpain I) with resultantmyofibril proteolysis has been suggested to underscore reperfusioninjury, as has proteolysis of troponin.

Reperfusion of tissue cells subjected to an injury have an alteredcellular metabolism, which in turn may contribute to delayed functionalrecovery. For example, an injury may induce anaerobic metabolism in thecell with a net production of lactate. Lactate release persists duringreperfusion, suggesting a delayed recovery of normal aerobic metabolism.Likewise, the activity of mitochondrial pyruvate dehydrogenase (PDH) maybe inhibited up to 40% after an injury and may remain depressed for upto 30 minutes after reperfusion.

Each of these events during reperfusion can lead to stress to the tissuecells and programmed cell death (apoptosis) and necrosis of the tissuecells. Apoptosis normally functions to “clean” tissues from wounded andgenetically damaged cells, while cytokines serve to mobilize the defensesystem of the organism against the pathogen. However, under conditionsof severe injury both stress response mechanisms can by themselves actas causes of death.

a. Flagellin

The flagellin may be a flagellin-related polypeptide. The flagellin maybe from any source, including a variety of Gram-positive andGram-negative bacterial species. Flagellin may have the amino acidsequence of one of 23 flagellins from bacterial species that aredepicted in FIG. 7 of U.S. Patent Publication No. 2003/0044429, thecontents of which are incorporated herein by reference. The nucleotidesequences encoding the flagellin polypeptides listed in FIG. 7 of U.S.2003/0044429 are publicly available at sources including the NCBIGenbank database.

Flagellin may be the major component of bacterial flagellum. Flagellinmay be composed of three domains (FIG. 10). Domain 1 (D1) and domain 2(D2) may be discontinuous and may be formed when residues in the aminoterminus and carboxy terminus are juxtaposed by the formation of ahairpin structure. The amino and carboxy terminus comprising the D1 andD2 domains may be most conserved, whereas the middle hypervariabledomain (D3) may be highly variable. Studies with a recombinant proteincontaining the amino D1 and D2 and carboxyl D1 and D2 separated by anEscherichia coli hinge (ND1-2/ECH/CD2) indicate that D1 and D2 may bebioactive when coupled to an ECH element. This chimera, but not thehinge alone, may includee IkBa degradation, NF-kB activation, and NO andIL-8 production in two intestinal epithelial cell lines. Thenon-conserved D3 domain may be on the surface of the flagellar filamentand may contain the major antigenic epitopes. The potent proinflammatoryactivity of flagellin may reside in the highly conserved N and C D1 andD2 regions.

Flagellin may induce NF-kB activity by binding to Toll-like receptor 5(TLR5). The TLR family may be composed of at least 10 members and isessential in innate immune defense against pathogens. The innate immunesystem may recognize pathogen-associated molecular patterns (PAMPs) thatare conserved on microbial pathogens. TLR may recognize a conservedstructure that is particular to bacterial flagellin. The conservedstructure may be composed of a large group of residues that are somewhatpermissive to variation in amino acid content. Smith et al., Nat.Immunol. 4:1247-53 (2003) have identified 13 conserved amino acids inflagellin that are part of the conserved structure recognized by TLR5.The 13 conserved amino acids of flagellin that may be important for TLR5activity are shown in FIG. 11.

The flagellin may be from a species of Salmonella, a representativeexample of which is S. dublin (encoded by GenBank Accession NumberM84972) (SEQ ID NO: 1). The flagellin related-polypeptide may be afragment, variant, analog, homolog, or derivative of SEQ ID NO: 1, orcombination thereof, that binds to TLR5 and induces TLR5-mediatedactivity, such as activation of NF-kB activity. A fragment, variant,analog, homolog, or derivative of flagellin may be obtained byrational-based design based on the domain structure of Flagellin and theconserved structure recognized by TLR5.

The flagellin may comprise at least 10, 11, 12, or 13 of the 13conserved amino acids shown in FIG. 11 (positions 89, 90, 91, 95, 98,101, 115, 422, 423, 426, 431, 436 and 452). The flagellin may be atleast 30-99% identical to amino acids 1 174 and 418 505 of SEQ ID NO: 1.FIG. 26 lists the percentage identity of the amino- and carboxy-terminusof flagellin with known TLR-5 stimulating activity, as compared to SEQID NO: 1.

The flagellin may be a flagellin polypeptide from any Gram-positive orGram-negative bacterial species including, but not limited to, theflagellin polypeptides disclosed in U.S. Pat. Pub. 2003/000044429, thecontents of which are incorporated herein, and the flagellin peptidescorresponding to the Accession numbers listed in the BLAST results shownin FIG. 25 of U.S. Patent Pub. 2003/000044429, or variants thereof.

The flagellin may stimulate TLR5 activity. Numerous deletional mutantsof flagellin have been made that retain at least some TLR5 stimulatingactivity. The flagellin may be a deletional mutant disclosed in theExamples herein, and may comprise a sequence translated from GenBankAccession number D13689 missing amino acids 185-306 or 444-492, or fromGenBank Accession number M84973 missing amino acids 179-415, or avariant thereof.

The flagellin may comprise transposon insertions and changes to thevariable D3 domain. The D3 domain may be substituted in part, or inwhole, with a hinge or linker polypeptide that allows the D1 and D2domains to properly fold such that the variant stimulates TLR5 activity.The variant hinge elements may be found in the E. coli MukB protein andmay have a sequence as set forth in SEQ ID NOS: 3 and 4, or a variantthereof.

Other agents may be used to target TLR5 receptors. These agents may beagonists of TLR5 and stimulate TLR5 activity. The agonist may be ananti-TLR5 antibody or other small molecule.

b. Injury

The effects of reperfusion may be caused by an injury to the bodycomponent. The injury may be due to ischemia, hypoxia, an infarction, oran embolism. Treatment of the injury may lead to reperfusion and furtherdamage to the body component.

(1) Ischemia

Ischemia may be an absolute or relative shortage of blood supply to abody component. Relative shortage may be a mismatch, however small, ofblood supplied (oxygen delivery) to a body component vs. blood requiredto a body component for the adequate oxygenation. Ischemia may also bean inadequate flow of blood to a part of the body due to a constrictionor blockage of blood vessels supplying it and may affect any bodycomponent in the body. Insufficient blood supply causes body componentsto become hypoxic, or, if no oxygen is supplied at all, anoxic. This maycause necrosis. The mechanisms of ischemia may vary greatly. Forexample, ischemia to any body component may be due to tachycardia(abnormally rapid beating of the heart), atherosclerosis (lipid-ladenplaque obstructing the lumen of arteries), hypotension (low bloodpressure in septic shock, heart failure), thromboembolisms (bloodclots), outside compression of blood vessels (tumor), embolisms (foreignbodies in the circulation, e.g., amniotic fluid embolism), sickle celldisease (abnormally shaped hemoglobin), infarctions, induced g-forceswhich restrict the blood flow and force the blood to extremities of thebody, localized extreme cold due to frostbite, ice, improper coldcompression therapy, and any other force that restricts blood flow tothe extremities such as a tourniquet. Force to restrict blood flow toextremities may be required due to severe lacerations, incisions,puncture such as a knifing, crushing injuries due to blunt force trauma,and ballistic trauma due to gunshot or shrapnel wounds. Ischemia may bea feature of heart diseases, ischemic colitis, transient ischemiaattacks, cerebrovascular accidents, acute renal injury, rupturedarteriovenous malformations, and peripheral artery occlusive disease.

(2) Hypoxia

Hypoxia may be a deprivation of adequate supply of oxygen. Hypoxia maybe pathological condition in which the body as a whole (generalizedhypoxia) or region of the body (tissue hypoxia) is deprived of adequateoxygen supply. A variation in levels of arterial oxygen may be due to amismatch between supply and demand of oxygen by body components. Acomplete deprivation of oxygen supply is anoxia. Hypoxia may behypoxemic hypoxia, anemic hypoxia, hypoxemic hypoxia, histotoxichypoxia, histotoxic hypoxia, and ischemic hypoxia.

Hypoxemic hypoxia may be an inadequate supply of oxygen to the body as awhole caused by low partial pressure of oxygen in arterial blood.Hypoxemic hypoxia may be due to low partial pressure of atmosphericoxygen such as at high altitudes, replacement of oxygen in breathing mixof a modified atmosphere such as a sewer, replacement of oxygenintentionally as in recreational use of nitrous oxide, a decrease inoxygen saturation of the blood due to sleep apnea, or hypopnea,inadequate pulmonary ventilation such as chronic obstructive pulmonarydisease or respiratory arrest, anatomical or mechanical shunts in thepulmonary circulation or a right to left shunt in the heart and lung.Shunts may cause collapsed alveoli that are still perfused or a block inventilation to an area of the lung. Shunts may present blood meant forthe pulmonary system to not be ventilated and prevent gas exchangebecause Thebesia vessels empty into the left ventricle and the bronchialcirculation, which supplies the bronchi with oxygen.

Anemia hypoxia may be the total oxygen content is reduced but thearterial oxygen pressure is normal. Hypoxemic hypoxia may be when bloodfails to deliver oxygen to target body components. Hypoxemic hypoxia maybe caused by carbon monoxide poisoning which inhibits the ability ofhaemoglobin to release the oxygen bound to it, or methaemoglobinaemia,an abnormal haemoglobin that accumulates in the blood.

Histotoxic hypoxia may be due to being unable to effectively use oxygendue to disabled oxidative phosphorylation enzymes.

(3) Infarction

Infarction is a is a type of pathological condition that can causeischemia. Infarction may be a macroscopic area of necrotic tissue causedthe loss of an adequate blood supply due to an occlusion. The infarctionmay be a white infarction composed of platelets and causes necrosis inorgan tissues such as heart, spleen, and kidneys. The infarction may bea red infarction composed of red blood cells and fibrin strands in organtissues of the lung. Disease associated with infarction may includemyocardial infarction, pulmonary embolism, cerebrovascular accident(stroke), acute renal failure, peripheral artery occlusive disease(example being gangrene), antiphospholipid syndrome, sepsis, giant cellarthritis, hernia, and volvulus.

(4) Embolism

Embolism is a type of pathological condition that can cause ischemia.Embolism may be an object that migrates from one part of the body andcauses an occlusion or blockage of a blood vessel in another part of thebody. An embolism may be thromboembolism, fat embolism, air embolism,septic embolism, tissue embolism, foreign body embolism, amniotic fluidembolism. Thromboembolism may be a blood clot that is completely orpartially detached from the site of thrombosis. Fat embolism may beendogenous fat tissues that escape into the blood circulation. Thefracture of bones is one example of a leakage of fat tissue into theruptured vessels and arteries. Air embolism may be a rupture of alveoliand inhaled air that leaks into the blood vessels. The puncture of thesubclavian vein or intravenous therapy are examples of leakage of airinto the blood vessels. A gas embolism may be gasses such as nitrogenand helium because insoluble and forming small bubbles in the blood.

c. Body Component

This invention relates to treatment of a body component in a mammal. Thebody component may be an organ, a tissue, or a cell. The body componentmay be from an abdomen, acetabulum, adipose, adrenal cortex, adrenalgland, adrenal medulla, alveolar macrophage, amnion, aorta, artery,ascites, ascitic fluid, axilla lymph node, bladder, blood, bone, bonemarrow, bowel, brain, breast, bronchus, cartilage, caudal trunk,cerebellum, cervix, chorionic villi, colon, conjunctiva, connectivetissue, cornea, dermis, dorsal root ganglion, duodenum, dysplastictongue mucosa, egg, embryo, endocrine, endometrium, endothelium,epidermis, epithelium, erythropoietic, eye, fibroblast, fin, foetus,foot, foreskin, Gasser's node, gingival stroma, gonad, groin lymph node,heart, humerus, ileum, intestine, ileocecal, ileum, islets ofLangerhanm, kidney, larvae, larval, larynx, liver, lung, lung(bronchioalveolar), lymph, lymph node, lymphatic tissue, lymphoid,lymphoid organs, mammary, mammary alveolar nodules, mammary gland,mesonephros, mesothelium, moulting nymph, mouth, muscle, nasal, nasalseptum, nervous system, neural, oesophageal gastric junction,oesophagus, oral, ovary, palatal mesenchyme, pancreas, papillaryovarian, penis, peripheral blood, peritoneum, pharynx, pituitary,placenta, pleural effusion, pleural fluid, prostate, pupal ovary,rectum, retina, right axial lymph node, salivary duct, sialaden,skeletal muscle, skin, small bowel, small intestine, soft tissue,spleen, sternum, stomach, tail, testicle, testis, thigh, thymus,thyroid, thyroid glands, tongue, tonsil, trachea, trunk, turbinate,umbilical cord, umbilicus, uterus, vagina, viscera, vulva, GI tract,lungs, kidneys, liver, cardiovascular system, blood vessel endothelium,central and peripheral nervous system, muscle, bone, hair follicles, andyolk sac.

3. COMPOSITION

This invention also relates to a composition comprising atherapeutically effective amount of flagellin. The composition may be apharmaceutical composition, which may be produced using methods wellknown in the art. The composition may also comprise a coagent. Asdescribed above, the composition may be administered to a mammal fortreating the effects of reperfusion.

a. Administration

Administration of the compositions using the method described herein maybe orally, parenterally, sublingually, transdermally, rectally,transmucosally, topically, via inhalation, via buccal administration, orcombinations thereof. Parenteral administration includes, but is notlimited to, intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, and intraarticular. For veterinary use, thecomposition may be administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal. The compositions may beadministered to a human patient, cat, dog, large animal, or an avian.

The composition may be administered simultaneously or metronomicallywith other treatments. The term “simultaneous” or “simultaneously” asused herein, means that the composition and other treatment beadministered within 48 hours, preferably 24 hours, more preferably 12hours, yet more preferably 6 hours, and most preferably 3 hours or less,of each other. The term “metronomically” as used herein means theadministration of the composition at times different from the othertreatment and at a certain frequency relative to repeat administration.

The composition may be administered at any point prior to reperfusionincluding about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr,106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr,6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. prior toreperfusion. The composition may be administered at any point prior tothe injury including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr,90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins,8 mins, 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.prior to injury.

The composition may be administered at any point after reperfusionincluding about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr,24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104hr, 106 hr, 108 hr, 110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hrafter reperfusion.

b. Formulation

The method may comprise administering a composition to treat for theeffects of reperfusion. Compositions provided herein may be in the formof tablets or lozenges formulated in a conventional manner. For example,tablets and capsules for oral administration may contain conventionalexcipients including, but not limited to, binding agents, fillers,lubricants, disintegrants and wetting agents. Binding agents include,but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth,mucilage of starch and polyvinylpyrrolidone. Fillers include, but arenot limited to, lactose, sugar, microcrystalline cellulose, maizestarch,calcium phosphate, and sorbitol. Lubricants include, but are not limitedto, magnesium stearate, stearic acid, talc, polyethylene glycol, andsilica. Disintegrants include, but are not limited to, potato starch andsodium starch glycollate. Wetting agents include, but are not limitedto, sodium lauryl sulfate. Tablets may be coated according to methodswell known in the art.

Compositions provided herein may also be liquid formulations including,but not limited to, aqueous or oily suspensions, solutions, emulsions,syrups, and elixirs. The compositions may also be formulated as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may contain additives including, but notlimited to, suspending agents, emulsifying agents, nonaqueous vehiclesand preservatives. Suspending agent include, but are not limited to,sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin,hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel,and hydrogenated edible fats. Emulsifying agents include, but are notlimited to, lecithin, sorbitan monooleate, and acacia. Nonaqueousvehicles include, but are not limited to, edible oils, almond oil,fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions provided herein may also be formulated as suppositories,which may contain suppository bases including, but not limited to, cocoabutter or glycerides. Compositions provided herein may also beformulated for inhalation, which may be in a form including, but notlimited to, a solution, suspension, or emulsion that may be administeredas a dry powder or in the form of an aerosol using a propellant, such asdichlorodifluoromethane or trichlorofluoromethane. Compositions providedherein may also be formulated as transdermal formulations comprisingaqueous or nonaqueous vehicles including, but not limited to, creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

Compositions provided herein may also be formulated for parenteraladministration including, but not limited to, by injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The composition may also be providedin a powder form for reconstitution with a suitable vehicle including,but not limited to, sterile, pyrogen-free water.

Compositions provided herein may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The compositions may be formulated withsuitable polymeric or hydrophobic materials (as an emulsion in anacceptable oil, for example), ion exchange resins, or as sparinglysoluble derivatives (as a sparingly soluble salt, for example).

c. Dosage

The method may comprise administering a therapeutically effective amountof the composition to a patient in need thereof. The therapeuticallyeffective amount required for use in therapy varies with the nature ofthe condition being treated, the length of time desired to increasehematopoietic stem cells into the bloodstream, and the age/condition ofthe patient. In general, however, doses employed for adult humantreatment typically are in the range of 0.001 mg/kg to about 200 mg/kgper day. The dose may be about 1 μg/kg to about 100 μg/kg per day. Thedesired dose may be conveniently administered in a single dose, or asmultiple doses administered at appropriate intervals, for example astwo, three, four or more sub-doses per day. Multiple doses may bedesired, or required.

The dosage may be at any dosage including, but not limited to, about 0.1μg/kg, 0.2 μg/kg, 0.3 μg/kg, 0.4 μg/kg, 0.5 μg/kg, 0.6 μg/kg, 0.7 μg/kg,0.8 μg/kg, 0.9 μg/kg, 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg,125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg,450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg,775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925μg/kg, 950 μg/kg, 975 μg/kg or 1 mg/kg.

4. COAGENT

Flagellin or the composition may be coadministered with a coagent. Thecoagent may be any compound that slows or prevents the effects ofreperfusion. The coagent may be an antioxidant. The antioxidant may beable to slow and prevent the oxidation of other molecules, cells,tissues or organs. The antioxidant may be vitamin E, ascorbic acid,glutathione, lipoic acid, uric acid, carotenes such as β-carotene andretinol, vitamin E, and coenzyme Q, thiols such as cysteine, cysteamine,glutathione, and bilrubin, amifostine, and flavanoids.

The coagent may be a sodium-hydrogen antiport inhibitor. Injury andreperfusion may result in marked intracellular acidosis. Asodium-hydrogen antiport inhibitor may be used to reduce protonextrusion and prevent increases in Ca2+. A sodium-hydrogen inhibitor maybe cariporide.

The coagent may be insulin. Insulin may be used to stimulate PDHactivity and prevent inhibition of PDH activity after reperfusion.

The coagent may be adenosine. Adenosine may be used to openmitochondrial KATP channels.

5. COMBINATION TREATMENT

The method may be used in combination with other methods to treat theinjury. The other methods may be treatments of myocardial infarction(heart attack), pulmonary embolism, cerebrovascular accident (stroke),peripheral artery occlusive disease (example being gangrene),antiphospholipid syndrome, sepsis, giant cell arteritis, hernia,volvulus, solid tumor cancers, decompression sickness, sickle cellanemia, puncture of the subclavian vein, bone fractures, high altitudesickness, recreational use of nitrous oxide, sleep apnea, hypopnea,shunts, anemia, carbon monoxide poisoning, methaemoglobinaemia,thromboembolism, fat embolism, air embolism, septic embolism, tissueembolism, foreign body embolism, amniotic fluid embolism, inducedg-forces, and external pressure to prevent blood flow due to severecuts, castration, or mangling. The method may also be used incombination with methods of treating reperfusion injuries such asadministering low doses of hydrogen sulfate (H₂S), glisoden, or wheatglialin, or performing therapeutic hypothermia or aortic cross-clamping.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

Example 1 Dose-Dependent Protection of Flagellin on Renal Function[Flagellin May be a TLR5 Agonist]

At particular dosages, flagellin does not affect renal function. Thiseffect was demonstrated by measuring the levels of creatinine in theserum of mice after systemic administration of different dosages offlagellin. C57BL/6 mice were injected with either 0.01 μg, 0.5 μg, 1.0μg, or 5.0 μg of flagellin and levels of serum creatinine (mg/dl) weremonitored daily as shown in FIG. 1. Administration of 5 μg of flagellinresulted in increased serum concentrations that was evident within 24hours after administration. After 24 additional hours (48 hours total),the levels of creatinine peaked and then fell back to background levelsby 72 hours after administration and then began to slowly rise to lowlevels again (FIG. 1). In contrast, administration of 1 μg of flagellinalso induced a rise in serum creatinine levels, but this was onlydetected as a single peak after 48 hours and then fell to backgroundlevels by 72 hours after administration. Administration of 0.5 μg and0.1 μg did not induce any measurable increases in serum creatininelevels throughout the study period.

Example 2 Dose-Dependent Effect of Flagellin on Renal Function

At particular dosages, flagellin is capable of protecting renal tissueof a mammal from the effects of acute renal ischemia. This effect wasdemonstrated by administering flagellin to mice before imposition ofrenal ischemia and measuring survival following reperfusion of theischemic kidneys. Specifically, 30 minutes before being subjected to 45minutes of bilateral renal pedicle occlusion, groups of C57BL/6 micewere given either various doses of flagellin (0.01 μg, 0.5 μg, 1.0 μg,or 5.0 μg per body) in 400 μl of PBS or PBS alone (400 μl) viaintravenous administration. Survival of the mice, levels of serumcreatinine, and histopathology data were then collected.

a. Survival

Bilateral renal pedicle occlusion was performed in the mice aspreviously detailed. Mice were given 20 U (units/ml) sodium heparin viaintraperitoneal administration 20 minutes before surgery. The mice wereanesthetized with phenobarbital and kept warm under a 60-W light bulbuntil surgery. Under aseptic conditions, the abdominal cavity was openedwith a midline incision and the bilateral renal pedicle was occludednontraumatically with a microvascular clamp (World PrecisionInstruments, Sarasota, Fla.) and the wound was temporarily closed with4-0 silk suture. Mice were placed on a heat pad under a 60 Watt lightbulb and a sensor tip of the Traceable™ Certificate Memory MonitoringThermometer (Fisher Scientific) was placed into the abdominal cavity toensure temperature maintenance at 32° C. during the imposition of renalischemia. Kidneys were subjected to ischemia for 45 minutes. Afterremoval of the clamp, immediate and complete renal reperfusion wasconfirmed visually and the peritoneal cavity was closed. Sham-operatedmice were treated in an identical manner except for the bilateral clampof the renal pedicle.

In the control group given PBS without flagellin, 80% of the animalsexpired within 5 days following reperfusion of the ischemic kidneys (seeFIG. 2 a). All animals given 5 μg of flagellin before imposition ofrenal ischemia expired within 5 days following reperfusion. In contrast,all animals given either 1 or 0.5 μg of flagellin before renal ischemiasurvived more than 45 days after reperfusion. The protective effect offlagellin in acute renal ischemia was also observed to be dose-dependentin that animals given 0.1 or 0.01 μg were not protected against theinjury.

b. Renal Function Measurement

Serum creatinine levels were also measured to determine the protectiveeffect of flagellin on renal function. Sham operated mice and micesubjected to bilateral renal I/R injury were anesthetized withisofluorane and bled from the postorbital plexus using a heparin-coatedmicrocapillary tube at 24-hour intervals. The serum was stored at −80°C. until measurement. Serum creatinine levels were measured using theCreatinine Kit (Sigma Diagnostics, Inc., St. Louis, Mo.). The protectiveeffect of flagellin was reflected by the low levels of serum creatininedetermined at 24 hours post-reperfusion in animals given 1.25 or 0.5 μgflagellin 30 minutes before imposition of ischemia (see FIG. 2 b).Animals given the non-protective low doses of flagellin (0.1 and 0.01μg) had higher levels of creatinine levels that fell just below thoseobserved in the control group that received PBS 30 minutes beforebilateral pedicle occlusion.

c. Histology Studies of Renal Tissue

High serum creatinine levels, an indication of renal dysfunction inducedby imposition of ischemia-reperfusion injury, were supported by thehistopathology of the ischemic kidneys 24 hours after reperfusion (seeFIG. 3). For immunohistochemistry, retrieved kidneys were halved,embedded in OCT compound (Sakura Finetek U.S.A., Torrence, Calif.), andimmediately frozen in liquid nitrogen. Coronal sections were cut (7 mm),mounted onto slides, dried for 1 hr, and then fixed in acetone for 10minutes. Slides were immersed in PBS for 10 min and in 3% hydrogenperoxide/methanol for 5 minutes at room temperature to eliminateendogenous peroxidase activity. Endogenous biotin activity was blockedwith the Biotin Blocking System (DAKO, Carpentaria, Calif.). Aftertreating with normal rat serum (1:100), anti-mouse Gr-1 mAb (RB6.8C5)diluted at 1:100 in PBS with 1% bovine serum albumin (BSA) to detectneutrophils, or 1:50 dilutions of rat anti-mouse CD4 mAb (GK1.5) todetect CD4+ T cells, rat anti-mouse CD8a mAb (53-6.7) to detect CD8+ Tcells, or rat anti-mouse macrophage (F4/80) mAb (SEROTEC, Raleigh, N.C.)was added to the sections. Control slides were incubated with rat IgG.After 1 hr, slides were washed 3× with PBS and incubated for 20 min withbiotinylated rabbit anti-rat IgG antiserum (Sigma Aldrich) diluted 1:100in PBS/1% BSA. After 3 washes in PBS, slides were incubated withstreptavidin-horseradish peroxidase (DAKO) for 20 min. The DAB(3,3′-diaminobenzidine) substrate-chromagen solution (VectorLaboratories, Inc., Burlingame, Calif.) was applied to the slides for0.5-3 min. After rinsing in dH2O, slides were counterstained withhematoxylin, washed with dH2O, cover-slipped, and viewed by lightmicroscopy. Images were captured using Image Pro Plus (MediaCybernetics, Silver Spring, Md.).

To stain TLR5, 1 mg of anti-TLR5 mAb (ABR-Affinity BioReagents, Inc.,Golden, Colo.) was applied to slides and incubated for 1 hr at roomtemperature and after washing biotinylated goat anti-mouse IgG antibodydiluted 1:100 for 30 min at room temperature. After applying the DAB,the slides were washed with tap water, dipped for 3 sec in hematoxylinand then washed. The slides were dehydrated with increasingconcentrations of ethanol to 50% and then immersed in citrasolve twicefor 10 min each. The slides were washed with tap water, cover-slipped,and viewed by light microscopy.

The use of serum creatinine levels as an indication of renal dysfunctioninduced by imposition of ischemia-reperfusion injury was supported bythe histopathology of the ischemic kidneys 24 hours after reperfusion(see FIG. 3). Control group animals given PBS 30 min before impositionof renal ischemia had severe tubular necrosis with caste formationevident 24 hours after reperfusion. Consistent with the induction ofrenal dysfunction by administration of 5 μg of flagellin, there wasevidence of renal pathology 30 min after administration of 5 μg offlagellin without imposing ischemia and this increased in severityfollowing imposition of renal ischemia and reperfusion with obvioushemorrhage, thrombosis, and caste formation. In contrast, animals given0.5 μg flagellin 30 min before imposition of ischemia had low levels ofleukocytic infiltration 24 hours after reperfusion but the renalarchitecture appeared relatively normal. The low, non-protective dose,0.1 μg flagellin, did not rescue the renal pathology induced byischemia/reperfusion injury. When kidneys of surviving animals wereexamined at day 7 post-reperfusion, marked decreases in tubular necrosisand leukocytic infiltration as well as the absence of thrombosis andcase formation were observed in animals given 0.5 μg flagellin prior toimposition of renal ischemia. (see FIG. 4).

d. Neutrophil Infiltration to Damaged Renal Tissue

Since neutrophil infiltration and activation is a major contributor tothe tissue injury following renal ischemia-reperfusion, ischemic kidneyswere retrieved 9 and 24 hours after reperfusion from animals treatedwith PBS alone or with 0.5 μg flagellin before imposition of ischemiaand the levels of neutrophil infiltration was assessed byimmunohistochemical staining of prepared tissue sections.

To directly determine the number of neutrophils, macrophages, CD4+ Tcells and CD8+ T cells in ischemic kidneys during reperfusion, onequarter pieces of the retrieved kidney were cut and weighed. The kidneyswere incubated in RPMI 1640 culture medium with 2% fetal calf serum for1 hr and then were pushed through a 70 mm cell strainer using a syringeplunger. The cells were collected and the erythrocytes lysed using ACKLysing Buffer (GIBCO, Grand island, NY). After 2 washes, viable cellswere counted using Trypan blue exclusion. Aliquots of the cells werepreincubated with anti-CD16/CD32 Fc receptor antibody (BD Pharmingen,San Diego, Calif.) for 5 min to block nonspecific antibody binding andthen samples were incubated with FITC-conjugated anti-CD45 mAb as wellas PE-conjugated antibody to detect macrophages (F4/80) or CD8+ T cells(53-6.7) and APC-conjugated antibody to detect neutrophils (RB6.8C5) orCD4+ T cells (GK1.5) (all antibodies from BD Pharmingen) for 30 min at4° C. Cells were analyzed using two-color flow cytometry on aFACSCalibur (BD Biosciences, San Jose, Calif.). The forward scatter andFL1 (CD45+) channels were used to gate the leukocytes in the kidneytissue followed by analysis of the specific leukocyte populations. Foreach sample, 200,000 events were accumulated. The data were analyzedusing CellQuest software (BD Biosciences). Total numbers of eachleukocyte population were calculated by: (the total number of leukocytescounted)×(% of the leukocyte population counted in the CD45+ cells)/100.The data are reported as number of each leukocyte population/g kidneytissue from sham and I/R animals.

Marked decreases in neutrophil infiltration were observed 9 and 24 hoursafter reperfusion when animals were given 0.5 μg flagellin (see FIG. 5a). Direct quantitation of leukocytic infiltration into the ischemickidneys indicated that 0.5 ug flagellin reduced neutrophil infiltrationalmost to the levels observed in the sham-operated control animals (seeFIG. 5 b). Decreases in the number of CD4 and CD8 T cells andmacrophages were observed in ischemic kidneys 24 hours after reperfusionand administration of 0.5 μg of flagellin 30 minutes before ischemiadecreased the number of both CD4 and CD8 T cells further.

Example 3 Flagellin Condition Decreases Pro-Inflammatory CytokineExpression During Reperfusion of Ischemic Kidneys

This example demonstrates the critical role of flagellin preventingchemokines CXCL1/KC and CXCL2/KC in directing leukocyte infiltrationinto ischemic kidney tissues. Previous studies have indicated that peaklevels of the neutrophil chemoattractants CXCL1/KC and CXCL2/KC inischemic kidneys occurs at 9 hours post-reperfusion [REFERENCE]

To begin to investigate mechanisms underlying the decreased leukocyticinfiltration into ischemic kidneys when animals were conditioned with0.5 ug flagellin, kidneys were removed 9 and 24 hours after reperfusionand the mRNA and protein levels of neutrophil and macrophagechemoattractants were determined (FIG. 6 a). One-quarter pieces were cutfrom harvested kidneys and frozen in liquid nitrogen. Total tissue RNAwas extracted using RNeasy™ Mini Kit (QIAGEN, Valencia, Calif.) andreverse transcribed using the High-Capacity cDNA Archive Kit (AppliedBiosystems, Foster City, Calif.). Real time PCR was performed on a Prism7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.)with test KC/CXCL1, MIP-2/CXCL2 and MCP-1/CCL2 primers and Mrp132 usedas the control (Applied Biosystems, Foster City, Calif.).

Kidneys samples stored in liquid nitrogen were dissolved in 500 ml ofPBS with 0.01M EDTA and a proteinase inhibitor cocktail (10 mg/mlphenylmethyl solfonyl fluoride, 2 mg/ml aprotinin, 2 mg/ml leupeptin,100 mg/ml Pefabloc SC, and 100 mg/ml chymostatin), and then 1 ml of 1.5%Triton X-100 in PBS was added. After incubation with agitation for 1 hrat 4° C., samples were centrifuged, the supernatant was collected, andthe total protein concentration was determined using the BCATM ProteinAssay Kit (Pierce, Rockford, Ill.). KC/CXCL1, MIP-2/CXCL2 and MCP-1/CCL2concentrations were measured by sandwich ELISA using Quantikine M Kits(R&D Systems, Minneapolis, Minn.). To determine the activation ofneutrophils during reperfusion of ischemic kidneys, the concentration ofmyeloperoxidase (MPO) was measured using the Mouse MPO ELISA test kit(Cell Sciences, Canton, Mass.). Results are reported as concentration oftest protein per mg of total tissue protein±SD.

Preconditioning with protective doses of flagellin (1.25 or 0.5 ug)resulted in significant decreases in mRNA expression and protein levelsof the neutrophil chemoattractants CXCL1 and CXCL2 at 9 hourspost-reperfusion. Expression of CCL2 mRNA or protein levels were low atboth 9 and 24 hours after reperfusion and were not further influenced bypreconditioning with flagellin. In addition, mRNA levels of the acutephase proteins IL-1b and IL-6 but not TNFa were also decreased inischemic kidneys at 9 hours post-reperfusion in flagellin preconditionedanimals (FIG. 6 b).

Example 4 Protective Effect of Flagellin when Administered DuringReperfusion of Ischemic Kidneys

This example demonstrates that flagellin provides a protective effect toacute ischemic treated kidneys when given after the initiation ofreperfusion. As described above, bilateral renal pedicle occlusion wasperformed in the mice and serum creatinine levels were measured todetermine the protective effect of flagellin on renal function afterinitiation of reperfusion.

Specifically, groups of C57BL/6 mice were subjected to 45 minutes ofbilateral renal pedicle occlusion and were administered 0.5 μg offlagellin at various times following the removal of the renal clamps(See FIG. 7). Administration of flagellin 30 minutes before or within 30min after declamping rescued the viability of all mice subjected to theischemic injury. Flagellin administration 1 hour as well as at latertimes after initiation of reperfusion failed to rescue any of the micefrom the injury. The protective effect of administering the flagellin 30minutes before declamping or within 30 minutes following declamping wasreflected by the low levels of serum creatinine monitored 24 hours afterreperfusion of the ischemic kidneys (FIG. 7 b).

Example 5 Protective Effect of Flagellin Requires TLR5 Signaling onRenal Parenchymal Cells

This example demonstrates the target source of protective effect offlagellin treatment during reperfusion of tissue. As discussed inExamples 1-4, reperfusion studies were performed on ischemic kidneys.

Radiation-induced bone marrow reconstituted chimeras were generatedbetween wild-type C57BL/6 and B6.MyD88^(−/−) mice. Radiation-inducedbone marrow reconstituted chimeras were generated by cutting the tips offemurs and tibias from wild-type C57BL/6 and B6.MyD88^(−/−) mice andflushing with RPMI 1640 to collect the bone marrow cells. Bone marrowrecipient mice first received 1100 Rad g-irradiation and then 3 hourslater received 20×10⁶ bone marrow cells intravenously. Irradiated CD90.1recipients received bone marrow from congenic CD90.1 donors or viceversa. The reconstituted recipients received antibiotics (0.2 mg/mlsulfamethoxazole and 0.4 mg/ml trimethoprim) in the drinking water fromday 1 to 7 as prophylaxis. The recipients were allowed to recover for8-12 weeks and complete chimerism was confirmed by staining peripheralblood cells with FITC-conjugated 90.2 and PE-conjugated 90.1.

FIG. 8 shows that administration of 0.5 μg flagellin within 30 minutesof reperfusion of ischemic kidneys of wild-type C57BL/6 micereconstituted with wild-type bone marrow decreased CXCL1 and CXCL2 mRNAlevels. In MyD88^(−/−) recipients reconstituted with either MyD88^(−/−)or wild-type bone marrow, little CXCL1 and CXCL2 mRNA was induced duringreperfusion of ischemic kidneys and administration of flagellin duringreperfusion of these kidneys did not decrease the mRNA levels of thesechemokines. In contrast, wild-type recipients of bone marrow fromMyD88^(−/−) donors expressed high levels of CXCL1 and CXCL2 mRNA andthese levels were decreased by administration of flagellin duringreperfusion of the ischemic kidneys. This demonstrates that the targetof the flagellin was a parenchymal kidney cell rather than a leukocyte.

To investigate this further, renal sections from wild-type C57BL/6 andBALB/c mice were stained with anti-TLR5 antibody (FIG. 9 a). The cellsstaining positively were primarily cells in the vasculature and stainingwas not apparent on renal tubular cells or glomeruli. Kidney sectionsfrom Moth Eaten mice that have a genetic defect in the expression of TLR5 did not stain with the anti-TLR5 antibody. Expression levels of TLR5mRNA were low in kidneys prior to imposition of renalischemia/reperfusion but increased quickly during reperfusion ofischemic kidneys (FIG. 9 b).

Example 6 Protective Effect of Flagellin in Hind Limb Ischemia Model

The potential protective effects of CBLB502 in a mouse hind limbischemia model were investigated in a simulation of a tourniquet-inducedischemic injury. These studies originated from studies indicating thatCBLB502 given to mice subjected to bilateral renal pedicle occlusionattenuated ischemic injury and renal dysfunction including decreasedneutrophil chemoattractant production in response to reperfusion,decreased neutrophil infiltration into the ischemic kidney, andattenuation of rises in serum creatinine levels and loss of viability.The protectant could be given either before imposition of renal pedicleocclusion, or more importantly for clinical use, up to 30 min afterreperfusion of the ischemic kidney.

The tourniquet-induced injury was modeled by tightening a wide rubberband on the left hind limb of mice for 2-4 hours. After the ischemictime, the rubber band was loosened and removed. The animals recoveredfrom anesthesia but exhibited an inability to use the ischemic limb,which was dragged behind them for periods of up to 9 days. The ischemicinjury also included edema of the limb that was clearly visible and thatwas quantified by wet-dry weight measures and comparison with thecontralateral, non-ischemic hind limb, and induction of high levels ofproinflammatory cytokines including neutrophil chemoattractants andintense neutrophil infiltration into the ischemic limb. In addition,injection of Evan's blue dye indicated considerable amounts of vascularleak in the ischemic limb (data not shown).

Studying the protective effects of CBLB502, mice were again subjected toa tourniquet-induced injury by tightening a wide rubber band on the lefthind limb for 3 hours. After the ischemic time, the rubber bank wasloosened and removed. Fifteen minutes upon removal of the rubber bandand initiation of reperfusion, 0.5 μg of CBLB502 or vehicle (PBS) wasadministered intramuscularly into the left ischemic limb. Miceadministered the CBLB502 has a more rapid recovery of limb usage and, byday 14 posted reperfusion, had a measurable grip strength for theischemic limb of 10 GF. In contrast, mice administered only PBS 15minutes post reperfusion did not achieve this strength until day 21.Limbs given CBLB502 also had almost no evidence of edema 25 hours afterreperfusion as evidence by a wet/dry weight ratio of 2.5 vs. 3.4 for theischemic limb from mice given only vehicle at reperfusion (See FIG.14C). With regard to vascular leak, the CBLB501 administered mice had a7.4 μg Evan's Blue Dye per gram web weight limb tissue vs. 13.1 μg forvehicle administered mice, P<0.001) (See FIG. 14D). Finally, limbstreated with CBLB502 at reperfusion had significant decreases in tissueneutrophil and macrophage chemoattractant sCXCL2, CCL2, andmyeloperoxidase (P<0.05 for each assay). A hematoxylin/eosin stain wasperformed on the hind limb muscle on day 14 after reperfusion following3 hours of ischemia on mice treated with CBLB502 (FIG. 14A) or vehicle(FIG. 14B).

Injection of CBLB502 within 30 min of reperfusion also resulted indecreases in neutrophil chemoattractant production and neutrophilinfiltration into the ischemic limb, visible decreases in edema, andaccelerated recovery (day 4-6) of the use of the ischemic limbs.Histological examination also indicated greater muscle fiber bundlethickness in the ischemic limbs of animals treated with the protectant(data not shown).

These results will be further investigated through quantitativemeasurement of inflammation and limb dysfunction in animals subjected tohind limb ischemia with vs. without administration of the CBLB502protectant. This will include quantification of other proinflammatorycytokines, direct quantification of neutrophil infiltration,quantitation of muscle fiber bundle thickness and apoptosis of musclefibers, and magnitude and duration of edema.

The invention claimed is:
 1. A method of treating an injury to a mammal,comprising administering to the mammal a composition comprisingflagellin, wherein the injury is tourniquet-induced injury to a limb ofthe mammal, and wherein the flagellin comprises the sequence of SEQ IDNO:
 8. 2. The method of claim 1, wherein the tourniquet-induced injuryis associated with reperfusion.
 3. The method of claim 1, wherein thetourniquet-induced injury induces ischemia.
 4. The method of claim 1,wherein the method reduces neutrophil chemoattractant production in thelimb.
 5. The method of claim 1, wherein the method reduces neutrophilinfiltration in the limb.
 6. The method of claim 1, wherein the methodreduces edema in the limb.
 7. The method of claim 1, wherein the methodreduces inflammation in the limb.
 8. The method of claim 1, wherein themethod reduces apoptosis of ischemic cells in the limb.
 9. The method ofclaim 1, wherein the method enhances muscle fiber bundle thickness inthe limb.
 10. The method of claim 1, wherein the composition isadministered prior to, together with, or after the influx of oxygenresulting from reperfusion.
 11. The method of claim 1, wherein thecomposition is administered in combination with an antioxidant.
 12. Themethod of claim 11, wherein the antioxidant is selected from amifostineand vitamin E.
 13. The method of claim 1, wherein the composition isadministered by intramuscular injection.